Validation of a Method for Quantification of Lutein in Spinach Using High-Performance Liquid Chromatography: Interlaboratory Study.

BACKGROUND: Lutein is gaining attention as a strong antioxidant contained in foods. It accumulates in the human blood and retina, and is considered to play an important role in the body, especially in the eyes. OBJECTIVE: A method to determine the lutein content of raw spinach (Spinacia oleracea L.) was developed with the aim of its enactment as a Japanese agricultural standard (JAS) measurement method for components beneficial to human health. METHODS: An interlaboratory study was conducted to evaluate an analytical method for the determination of lutein in spinach. The detection limit and quantification limit of lutein for this method were 0.2 and 0.7 mg/kg, respectively. Twelve participating laboratories independently analyzed test samples (five pairs of blind duplicates) using high-performance liquid chromatography (HPLC). RESULTS: After removal of a few outliers, the repeatability relative standard deviation (RSDr), reproducibility (RSDR), and predicted RSDR of the evaluated method were 3.4-7.5, 4.6-13, and 7.5-8.5%, respectively, in a concentration range from 64.9-150 mg/kg. CONCLUSIONS: The HorRat values (RSDR/predicted RSDR) of the lutein concentration were calculated to be 0.61-1.6. HIGHLIGHTS: The study results indicate the acceptable precision of this method.

Determination of Lycopene Concentration in Fresh Tomatoes by Spectrophotometry: A Collaborative Study.

BACKGROUND: Lycopene has been the object of considerable research attention recently, and the effects of the intake of lycopene, or of tomato products, have been studied in various ways. In Japan, interest in the health-promoting function of food components has increased. OBJECTIVE: Developing a method to determine lycopene contents in tomato that meets the Japanese Agricultural Standard (JAS). METHOD: In the proposed JAS method, the test sample consists of fresh tomatoes; a hexane-acetone mixture is utilized as the extraction solvent. A collaborative study was conducted to evaluate the interlaboratory performance of the method. RESULTS: Ten laboratories participated and analyzed six test materials characterized by a lycopene content between 39 and 170 mg/kg as blind duplicates. After removing statistical outliers, RSDr ranged from 1.2 to 3.0% and RSDR ranged from 2.4 to 4.2%. The HorRat values were calculated and found to be in the 0.26-0.49 range. CONCLUSIONS: The method for determining the lycopene content in tomato was evaluated by means of a collaborative study, and the reproducibility of this method was found to be acceptable. HIGHLIGHTS: Intended for standardization in Japan, a method to determine lycopene content in tomato has been developed and shown to have acceptable precision in a collaborative study.

Improvement and Interlaboratory Validation of the Lipophilic Oxygen Radical Absorbance Capacity: Determination of Antioxidant Capacities of Lipophilic Antioxidant Solutions and Food Extracts.

A lipophilic oxygen radical absorbance capacity (L-ORAC) assay is an evaluation of the antioxidant capacity of solutions of lipophilic compounds. The concentrations of fluorescein, radical generator, and Trolox standard solutions were optimized to improve the precision of the assay. An interlaboratory study using two antioxidant solutions and three food extracts as test samples conducted in accordance with harmonized protocol demonstrated satisfactory L-ORAC measurements; the intermediate precision relative standard deviations (RSD(int)) ranged from 7.0 to 16.7%, the reproducibility relative standard deviations (RSD(R)) ranged from 14.8 to 19.4%, and the HorRat values ranged from 1.35 to 1.78.

Improvement of the lipophilic-oxygen radical absorbance capacity (L-ORAC) method and single-laboratory validation.

We improved the procedure for lipophilic-oxygen radical absorbance capacity (L-ORAC) measurement for better repeatability and intermediate precision. A sealing film was placed on the assay plate, and glass vials and microdispensers equipped with glass capillaries were used. The antioxidant capacities of food extracts can be evaluated by this method with nearly the same precision as antioxidant solutions.

Evaluation of a method to quantify quercetin aglycone in onion (Allium cepa) by single- and multi-laboratory validation studies.

Official Methods of Analysis of AOAC International (OMA) 2006.07 was originally designed for quantifying flavonol aglycones in ginkgo dietary supplements. To determine whether the method is applicable to the quantification of flavonol aglycones in lyophilized onion samples, single- and multi-laboratory validation studies were performed. Triplicated measurements on 3 different days revealed that the mean quercetin content was 3.48 g/kg dry weight, and the relative repeatability standard deviation (RSD(r)) and the relative intermediate standard deviation (RSD(int)) were 0.8 and 1.8%, respectively. The recovery of quercetin-3-O-glucoside spiked at 3 different amounts (1.56, 3.12, and 6.24 g/kg dry weight of onion) ranged from 98.42 to 100.31%, and the RSD(r) and RSD(int) ranged from 2.2 to 5.9%, and from 3.4 to 5.2%, respectively. A multi-laboratory validation study showed that the mean quercetin contents were 2.80 and 6.61 g/kg dry weight, and that satisfactory inter-laboratory precision (RSD(r) and RSD(R) ranged from 0.41 to 0.92%, and from 6.73 to 7.62%, respectively); all HorRat values were less than 2. These results indicate that OMA 2006.07 is applicable to the determination of the quercetin content of lyophilized onion samples.

Method validation by interlaboratory studies of improved hydrophilic oxygen radical absorbance capacity methods for the determination of antioxidant capacities of antioxidant solutions and food extracts.

Hydrophilic oxygen radical absorbance capacity (H-ORAC) is a method for evaluating antioxidant capacities of solutions of hydrophilic compounds. In this study, we improved the original method for H-ORAC determination, and evaluated the precision of the two improved methods (methods A and B) by interlaboratory studies using 5 antioxidant solutions and 5 food extracts as test samples. An interlaboratory study of method A, in accordance with the harmonized protocol, demonstrated satisfactory performance (intermediate precision relative standard deviations (RSD(int)) ranging from 4.6 to 18.8%; the reproducibility relative standard deviations (RSD(R)) ranging from 7.0 to 21.1%, and the HorRat values ranging from 0.40 to 1.93). However, methodological problems remained, and a further improved method, method B, was thus developed. An interlaboratory study of method B by 5 participating laboratories showed better intermediate precision and reproducibility (RSD(int) and RSD(R) ranging from 1.8 to 9.4%, and from 4.4 to 13.8%, respectively), and all HorRat values for the test samples were less than 1.3, suggesting good performance for the H-ORAC measurement.

The use of summary statistics for sample size allocation for food composition surveys and an application to the potato group.

To give a quantitative guide to sample size allocation for developing sampling designs for a food composition survey, we discuss sampling strategies that consider the importance of each food; namely, consumption or production, variability of composition, and the restrictions within the available resources for sample collection and analysis are considered., Here we consider two strategies: 'proportional' and 'Neyman' are discussed. Both of these incorporate consumed quantity of foods, and we review some available statistics for allocation issues. The Neyman optimal strategy allocates less sample size for starch than proportional, because the former incorporates variability in the composition. Those strategies improved accuracy in dietary nutrient intake more than equal sample size allocation. Those strategies will be useful as we often face sample size allocation problems, wherein we decide whether to sample 'five white potatoes and five taros or nine white and one taros'. Allocating sufficient sample size for important foodstuffs is essential in assuring data quality. Nevertheless, the food composition table should be as comprehensive as possible.

Determination of the geographic origin of onions between three main production areas in Japan and other countries by mineral composition.

Onions (Allium cepa L.) are produced in many countries and are one of the most popular vegetables in the world, thus leading to an enormous amount of international trade. It is currently important that a scientific technique be developed for determining geographic origin as a means to detect fraudulent labeling. We have therefore developed a technique based on mineral analysis and linear discriminant analysis (LDA). The onion samples used in this study were from Hokkaido, Hyogo, and Saga, which are the primary onion-growing areas in Japan, and those from countries that export onions to Japan (China, the United States, New Zealand, Thailand, Australia, and Chile). Of 309 samples, 108 were from Hokkaido, 52 were from Saga, 77 were from Hyogo, and 72 were from abroad. Fourteen elements (Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba) in the samples were determined by frame atomic adsorption spectrometry, inductively coupled plasma optical emission spectrometry, and inductively coupled plasma mass spectrometry. The models established by LDA were used to discriminate the geographic origin between Hokkaido and abroad, Hyogo and abroad, and Saga and abroad. Ten-fold cross-validations were conducted using these models. The discrimination accuracies obtained by cross-validation between Hokkaido and abroad were 100 and 86%, respectively. Those between Hyogo and abroad were 100 and 90%, respectively. Those between Saga and abroad were 98 and 90%, respectively. In addition, it was demonstrated that the fingerprint of an element pattern from a specific production area, which a crop receives, did not easily change by the variations of fertilization, crop year, variety, soil type, and production year if appropriate elements were chosen.

Determination of the acid value of instant noodles: interlaboratory study.

An interlaboratory study was performed to evaluate the method for determining the acid value of instant noodles, based on the Japanese Agricultural Standard (JAS), with extraction of lipid using petroleum ether at a volume of 100 mL to the test portion of 25 g. Thirteen laboratories participated and analyzed 5 test samples as blind duplicates. Statistical treatment revealed that the repeatability (RSDr) of acid value was <6.5%, and the reproducibility (RSDR) of acid value was <9.6%. The HorRat values (RSDR/predicted RSDR) were 1.2-1.8, where the RSDR and the predicted RSDR were obtained in terms of free fatty acids in the noodles per unit weight, using the equation [acid value = percent free fatty acids (as oleic) x 1.99] and the extracted lipid contents. This method was shown to have acceptable precision by the present study.

Effects of fertilization, crop year, variety, and provenance factors on mineral concentrations in onions.

Mineral concentrations of onions (Allium cepa L.) grown under various conditions, including factors (fertilization, crop year, variety, and provenance), were investigated to clarify how much each factor contributes to the variation of their concentrations. This was because the mineral concentrations might be affected by various factors. The ultimate goal of this study was to develop a technique to determine the geographic origins of onions by mineral composition. Samples were onions grown under various conditions at 52 fields in 18 farms in Hokkaido, Japan. Twenty-six elements (Li, Na, Mg, Al, P, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Rb, Sr, Y, Mo, Cd, Cs, Ba, La, Ce, Nd, Gd, W, and Tl) in these samples were determined by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. Fertilization conditions and crop years of onions caused variations of P, Ni, Cu, Rb, Sr, Mo, Cs, and Tl concentrations in onions; different onion varieties also showed variations in numerous element concentrations. However, the variations of mineral compositions of onions by these factors were smaller than the differences between production places with a few exceptions. Furthermore, Na, Rb, and Cs in group IA of the periodic table, Ca, Sr, and Ba in group IIA, and Zn and Cd in group IIB showed similar concentration patterns by group; this result demonstrated that elements in the same periodic groups behaved similarly in terms of their absorption in onions.

Determination of the moisture content of instant noodles: interlaboratory study.

Determination of the moisture content of instant noodles, currently under discussion by the Codex Alimentarius Commission (CAC) requires 2 methods: one for fried noodles and the other for nonfried noodles. The method to determine the moisture content of fried noodles by drying at 105 degrees C for 2 h used in the Japanese Agricultural Standard (JAS) system of Japan can be applied to this purpose. In the present study, the JAS method for fried noodles was modified to be suitable for nonfried noodles by extending the drying time to 4 h. An interlaboratory study was conducted to evaluate interlaboratory performance statistics for these 2 methods. Ten participating laboratories each analyzed 5 test materials of fried and nonfried noodles as blind duplicates. After removal of outliers statistically, the repeatability (RSDr) and the reproducibility (RSD(R)) of these methods were 1.6-2.6 and 3.9-4.8% for fried noodles, and 0.3-1.5 and 1.3-2.9% for nonfried noodles, respectively.

Method performance study of the determination of total nitrogen in soy sauce by the Kjeldahl method.

The objective of this collaborative study was to evaluate the proposed method for determining the total nitrogen in soy sauce by the Kjeldahl method submitted to the Codex Alimentarius Commission for endorsement in accordance with the protocol for the design, conduct, and interpretation of method-performance studies. The digestive conditions of the proposed method are the addition of 10 mL of H2SO4, 10 g (8 g by using a block digester) of K2SO4, and 1 mL of 20% CuSO4 x 5H2O and 80 min boiling period after the liquid is cleared by a heating device. Seventeen laboratories participated, analyzing five soy sauce samples as blind duplicates. Since the volume sampling method used in the JAS (Japanese Agricultural Standard) method showed lower accuracy of data because of the density of soy sauce, the method of sampling by weight was adopted as the proposed method. The total amount of outlier data was within acceptable limits for method-performance studies (< or = 22.2%). Lysine and ammonium sulfate recoveries for all laboratories were > or = 98% and > or = 99% respectively. The RSDr (repeatability relative standard deviation) values ranged from 0.4 to 1.3%, and the RSDR (reproducibility relative standard deviation) values were from 0.8 to 1.9%. HORRAT (RSDR/predicted RSDR) for the reproducibility showed 0.2 to 0.4, indicating acceptable precision of the method and excellent analytical performance.

Application of inorganic element ratios to chemometrics for determination of the geographic origin of welsh onions.

The composition of concentration ratios of 19 inorganic elements to Mg (hereinafter referred to as 19-element/Mg composition) was applied to chemometric techniques to determine the geographic origin (Japan or China) of Welsh onions (Allium fistulosum L.). Using a composition of element ratios has the advantage of simplified sample preparation, and it was possible to determine the geographic origin of a Welsh onion within 2 days. The classical technique based on 20 element concentrations was also used along with the new simpler one based on 19 elements/Mg in order to validate the new technique. Twenty elements, Na, P, K, Ca, Mg, Mn, Fe, Cu, Zn, Sr, Ba, Co, Ni, Rb, Mo, Cd, Cs, La, Ce, and Tl, in 244 Welsh onion samples were analyzed by flame atomic absorption spectroscopy, inductively coupled plasma atomic emission spectrometry, and inductively coupled plasma mass spectrometry. Linear discriminant analysis (LDA) on 20-element concentrations and 19-element/Mg composition was applied to these analytical data, and soft independent modeling of class analogy (SIMCA) on 19-element/Mg composition was applied to these analytical data. The results showed that techniques based on 19-element/Mg composition were effective. LDA, based on 19-element/Mg composition for classification of samples from Japan and from Shandong, Shanghai, and Fujian in China, classified 101 samples used for modeling 97% correctly and predicted another 119 samples excluding 24 nonauthentic samples 93% correctly. In discriminations by 10 times of SIMCA based on 19-element/Mg composition modeled using 101 samples, 220 samples from known production areas including samples used for modeling and excluding 24 nonauthentic samples were predicted 92% correctly.

Chemometric techniques on inorganic elements composition for the determination of the geographic origin of Welsh onions.

Techniques to determine the producing country of Welsh onions (Allium fistulosum L.) were developed by using 216 samples from Japan and from Anchu Shandgon, Shanghai and Amoy Fjian in China. Twelve elements (Na, P, K, Ca, Mg. Mn, Fe, Cu, Zn, Sr, Ba, Al, Co, Ni, Rb, Mo, Cd, Cs, La, Ce, Tl and Pb) contained in Welsh onions were determined. In order to determining whether Welsh onions originate in Japan or in China, linear discriminant analysis (LDA) and soft independent modeling of class analogy (SIMCA) were performed, using concentrations of 20 elements excluding Al and Pb in 22 elements. In LDA for two-group discrimination modeled on Welsh onions from Japan and China, 103 samples used for modeling were classified 95% correctly, and 89 other samples were predicted 94% correctly. As the average of predictions by SIMCA modeled on samples selected by 10 times of random selections, 192 samples, including the 103 samples used for modeling, were predicted 96% correctly. In discriminations by combined LDA and SIMCA, all 81 samples produced in Japan were correctly judged to be Japanese ones, and only 8 samples out of 111 samples produced in China were not correctly judged to be Chinese ones.

[Changes in the cadmium content of rice during the milling process].

Milled rice and bran were prepared from brown rice (Oryza sativa japonica) using a commercial scale rice-mill to investigate the changes in cadmium (Cd) content caused by the milling process. Rice sample solution was prepared by microwave digestion using HNO3 and HF, then analyzed by ICP-MS. Cd was measured at m/z 114 in interference correction with MoO and Sn. The digestion method was validated with 4 kinds of certified reference material of "rice flour". We found that the Cd content was reduced only slightly by the milling process. When the Cd concentration of brown rice was regarded as 100, the relative content of the milled rice was 97 (average of 6 samples). In addition, the reduction of the Cd content was only slight when milled rice was processed to "wash-free" rice. The relative Cd content of the bran was 139 when that of brown rice was regarded as 100. These results support the hypothesis that the Cd content is not greatly decreased by the milling process.